Product development – grape craft beer

Telmo Gabriel Barros Nunes Product Development – Grape Craft Beer Master’s Degree Dissertation Master’s Degree in Food Science and Technology Work carried out under the guidance of Professor José Maria Marques Oliveira October 2022 ii Telmo Gabriel Barros Nunes Product Development – Grape Craft Beer Master’s Degree Dissertation Master’s Degree in Food Science and Technology Work carried out under the guidance of Professor José Maria Marques Oliveira October 2022 Supervisor: João Pedro Fernandes iii Copyright and working conditions by third parties This is an academic work that may be used by third parties, provided that the international rules and good practices accepted in respect of copyright and related rights are respected. Thus, the present work can be developed in terms of public availability. If the user needs to be authorized to use this work in conditions not subject to prior agreement, contact the author through the University of Minho's RepositóriUM. iv Acknowledgments With the dissertation and internship period complete, I would first like to thank my supervisor, José Oliveira, and supervisor at the company of internship, João Fernandes, for all the help and support they gave, as well as all the patience and availability with which they answered my many doubts and questions. Furthermore, I would like to thank everyone in Sovina Lda. for the knowledge and support they provided and the open arms with which I was received. Lastly, a special thanks to my family and close friends for the never-ending support and motivation they provided, with which I could not have completed this internship. v Statement of integrity I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. vi Product Development – Grape Craft Beer Abstract The craft beer market in Portugal is growing, even though it represents a small percentage of national beer production. This is a very competitive market, resulting from a growing demand for craft beer and the innovative character that is already inherent to this industry, thus putting pressure on companies to have a constant launch of new quality and outstanding products. It is therefore necessary to invest in the innovation and development of products that stand out from industrial beer, in order to draw the population's attention to this particular product and market. Innovation in this industry can be achieved by the incorporation of vinic characteristics on beer (through the use of grape must), since even though these two products share similarities on their production process, they vary dramatically in their social context, so it can be beneficial to integrate positive characteristics of wine in beer, or vice versa . Through the work carried out on this study, a bottom fermentation was defined as ideal for the envisioned product, where there is a noticeable presence of the grape must used (white grape noble variety Loureiro), which was verified and confirmed by the acceptance test performed, that served as an indicator of the potential viability of this product on the market. Other characteristics such as malted and unmalted cereals, hops and guidelines for the process (such as an industrial plan and HACCP analysis) were also defined, with lager beer malts, flaked oats and citric hops conferring the best sensory profile to the final product. However, this study would beneficiate from future testing to improve the formula here used, as careful planning around the quality of the grape must and environment in which the yeast operates is essential for the final product quality. Keywords: Craft Beer, Fermentation, Grape Ale, Grape Lager. vii Desenvolvimento de Produto – Cerveja Artesanal de Uva Resumo O mercado da cerveja artesanal em Portugal está em crescimento, ainda que represente uma pequena percentagem da produção de cerveja nacional. Este é um mercado bastante competitivo, o que resulta de uma procura cada vez maior por cerveja artesanal e pelo caráter inovador que já é inerente a esta indústria, colocando assim pressão nas empresas para terem um lançamento constante de novos produtos de qualidade e destaque. Assim, é necessário apostar então, na inovação e desenvolvimento de produtos que se destaquem em relação à cerveja industrial, de modo a chamar a atenção da população para este produto e mercado em particular. Inovação nesta indústria pode ser conseguida através da incorporação de características vínicas no mundo da cerveja (através de mosto de uva), já que apesar de se tratarem de produtos com semelhanças ao longo do seu processo de produção, variam drasticamente no seu contexto social, daí poder ser benéfico integrar características positivas do vinho na cerveja, ou vice-versa. Através do trabalho realizado neste estudo, definiu-se uma baixa fermentação como ideal para o produto pretendido, onde existe uma presença mais notória do mosto de uva utilizado (uva branca, casta Loureiro), o que foi verificado e confirmado pelo teste de aceitação realizado, que serviu como um indicador da potencial viabilidade deste produto no mercado. Outras características como maltes, lúpulos e diretrizes para o processo (como um plano industrial e análise HACCP) também foram definidas, com maltes de cerveja lager, aveia e lúpulos cítricos a conferirem o melhor perfil sensorial ao produto final. Contudo, este estudo beneficiaria de testes futuros para melhorar a fórmula aqui utilizada, visto que planeamento cuidadoso à volta da qualidade do mosto de uva e do ambiente no qual a levedura vai operar são essenciais para garantir a qualidade do produto final. Palavras-chave: Cerveja Artesanal, Fermentação, Grape Ale, Grape Lager. viii Table of Contents Copyright and working conditions by third parties ................................................................................ iii Acknowledgments .............................................................................................................................. iv Statement of integrity ..........................................................................................................................v Abstract ............................................................................................................................................. vi Resumo ............................................................................................................................................ vii Index of Tables .................................................................................................................................... x Index of Figures.................................................................................................................................. xi 1. Introduction and objectives ......................................................................................................... 1 1.1. Objectives ........................................................................................................................... 1 1.2. Brewery Sovina ................................................................................................................... 1 1.3. Dissertation Organization .................................................................................................... 2 2. Theoretical Overview ................................................................................................................... 3 2.1. Drinks Industry ................................................................................................................... 3 2.2. Drinks Industry Future......................................................................................................... 3 2.3. Product Development ...................................................................................................... 4 2.4. Sensory Analysis ................................................................................................................. 8 2.5. Brewing Industry ................................................................................................................. 9 2.6. Beer as a Product ............................................................................................................. 10 2.6.1. Raw materials ........................................................................................................... 11 2.6.2. Process of production ................................................................................................ 17 2.7. Food Safety – Quality Control ............................................................................................ 19 2.8. Beer types ........................................................................................................................ 22 2.8.1. Grape Ales and Lagers .............................................................................................. 22 3. Materials and Methods ............................................................................................................. 25 3.1. Brewing and fermentation equipment ................................................................................ 26 3.2. Raw Materials ................................................................................................................... 26 3.2.1. Malts ........................................................................................................................ 26 3.2.2. Hops ......................................................................................................................... 27 3.2.3. Yeasts and Bacteria ................................................................................................... 27 3.2.4. Adjuncts .................................................................................................................... 27 3.2.5. Salts ......................................................................................................................... 27 3.3. Production Process ....................................................................................................... 27 3.4. Sensory Analysis ............................................................................................................... 28 ix 3.5. Statistical Analysis ............................................................................................................ 30 3.6. Industrial Plan................................................................................................................... 31 4. Results and Discussion ............................................................................................................. 32 4.1. Study on the Production of Grape-based Craft Beer............................................................ 32 4.1.1. First Product Test – Grape Ale ................................................................................... 32 4.1.2. Second Product Test – Grape Lager ........................................................................... 37 4.1.3. Third Product Test – Grape Weiss .............................................................................. 40 4.2. Culmination of the study -- Final Product Test ................................................................ 43 4.3. Sensory Comparison of the Beers Produced in the Product Tests ....................................... 46 4.4. Future Tests ..................................................................................................................... 48 4.5. Industrial Plan................................................................................................................... 49 5. Conclusions .............................................................................................................................. 58 References ....................................................................................................................................... 59 Appendices ...................................................................................................................................... 66 A – Raw Material Characteristics .................................................................................................. 66 B – Fermentation Profile of the Product Tests ............................................................................... 68 C – Statistical Analysis Calculations and Data ............................................................................... 70 D – Data on Global Appreciation of the Different Beers Produced for this Study ............................. 71 4 industry. With the increase in the purchasing power of consumers and the consequent increase in drinks industry products consumption, companies are betting on the development of new products (DNP) with greater added value. Globalization and the ease of communication and transport and the elimination of barriers to international trade imply a quicker spread of novelties. The industry should, therefore, continuously invest in research and development (R&D) of products with the desired characteristics, in order to guarantee their survival in the market. 2.3. Product Development The ability to innovate is one of the biggest competitive advantages a company could have, especially when it comes to a significant change in some product, service or process, and it brings considerable benefits to the company's economy (Penso, 2003). Innovation involves new product development, production processes, marketing methods or new strategies of organization and is the result of a complex process that takes new or significantly improved ideas to the market or simply incorporates new activities essential to the innovation process (OECD, 2005). Society, markets and products change at great speed and innovation and R&D of new products or the alteration of an existing product that can be presented to the consumer with a new name has a fundamental role in the differentiation, growth and profit of companies. The need for product development can come from an internal request (relevant legal and regulatory changes, international or national standards, codes of industrial practices, company's own needs) or an external request (customer and market needs and expectations) (OECD, 2005). New product development is a process in which the entity transforms market opportunities and technical possibilities into a commercial product, ranging from preliminary stages, such as identifying the need for this process, to final stages, such as launching and monitoring the product (Penso, 2003). However, this process requires investment in money and human resources on the part of the company and, therefore, extensive bibliographic research is needed about the product's potential, as well as planning the product's development, from its initial formulation to the final product. The objective of entities is that this process can occur as fast as possible and with the least investment, so that it becomes economically viable for the company. A lot of definitions are possible for new products, depending on their traits, however, Fuller (2016) categorizes them in 7 types, according to Table 1. The need to increase financial profit, required by business administration, is a result of the company's growth in the market. Thus, the export of products, the implementation of marketing strategies and the constant development and introduction of new products allow the company to remain competitive and 5 profitable at all times. The need for new products can also be a direct result of the consumer himself, as changes in choices, lifestyle, health status, age group, education and purchasing power heavily influence market demands (Penso, 2003). Table 1. Types of new products (adapted from Fuller, 2016) New Products Characteristics Extension of a line of products Development of a new variation of a product line. This type of product requires little research and only small changes in marketing strategy, with no need for changes to processing lines or equipment. This product also has the benefit of not requiring new raw materials, nor new storage and distribution methodology, however, it needs testing with consumers and public campaigns. New functions for existing products Attributing a new function (for example, health benefits) to existing products originates a type of new product, at the cost of a change in marketing and sales strategy (new label, packaging, advertising campaigns) in order to reach a new market niche. New version of existing products New versions of existing product, be it through changes in the process of production or new uses entirely. It involves a change of concept and for that, research and development are required. May involve changes in facilities and investment in equipment, and marketing and sales strategies require changes. Reformulation of products Reformulation of existing products, which could be through changes in colour, flavour, texture, appearance, caloric value, proteins, minerals, enhancing the physicochemical characteristics and/or organoleptic properties due to market needs, and reducing the cost of production of ingredients or packaging materials. The objective of this type of product is to reach new market niches and new consumption trends, requiring some research and development. New packaging Introduction of the same product with a new package which could aim at different goals: increasing the product's shelf life, making the packages recyclable, reducing costs, increasing practicality, adding value to the product, among others. Innovative products An innovative product can be the result of changes to existing products. Often requires the incorporation of new ingredients and investment in new technologies, with associated long development times and high research costs. Also requires new marketing and sales policies. Creative product Associated with rare products, that create a new niche in the market for themselves. They require long periods of research and development, as well as a high investment. May require completely new or even exclusive facilities and equipment. Requires an investment in marketing and sales policy. It may be necessary to create a new company or brand, and they have an associated high risk of failure as competitors are likely to quickly copy the new product at lower costs. The life cycle of products is increasingly reduced due to market competitiveness and rapid obsolescence of products, which increases the need for new product development. Every product that is launched on the market has a life cycle that translates the acceptance of the product by the customer (expressed in sales or in generated profit) (Fuller, 2016). Initially, the product is introduced to the market and sales grow slowly and, consequently, the profit generated does not yet compensate the investments in the R&D process and in the marketing strategy. Subsequently, sales increase, as customers repeat the purchase and/or recommend to new customers, until the profit from the sales exceeds the expenses. 6 Eventually a slowdown in sales happens due to the appearance of new competing products and a stabilization ensues, where there is a reduction in profits and new investments are needed to change the image of the packaging and/or marketing strategies, so that the product survives. When sales drop abruptly and there is no longer any profit, the product's permanence in the market becomes unfeasible and its discontinuity may be necessary (Fuller, 2016). For companies to survive in the market, the profit generated must last over time (Figure 1) and new products must be introduced sequentially on the market. Figure 1. New product development contribution to company profit (adapted from Fuller, 2016). New product development is challenging in the sense of the dynamism of the market, the process of understanding the expectations and desires of consumers, the details and processes that involve each step of development, the pressure of time and financial investment, and the decision-making process for launching the product. It is critical that business management ensures that this process is well managed and successful. Due to the complexity involved in this process, it is important to assure its effectiveness and ensure the product's success in the market. For that purpose, the Stage-Gate model was hypothesized by Cooper (Cooper, 1990 and 2008), which was later adopted by several companies around the world. It is a conceptual and operational model that aims to reduce the time involved in the process of developing new products, and that intends to guarantee the good acceptability of the product on the market (sales and profit), minimizing failures and ensuring efficiency and effectiveness of the process (de Mello et al. , 2012). This model starts with the generation of ideas and ends with the launch of the product and consists of stages and decision “gates”. At each stage, the development work is carried out where the necessary information is obtained and analysed, and with the progress of the five stages, the uncertainties and risks of the process are reduced, however, the costs increase and so does the commitment. Each step is carried out by a multidisciplinary team that covers different areas in the company (de Mello et al. , 2012). The decision “gates” serve as quality control, where decisions are taken in order to continue investing in P R O F I T Time 7 the project, and where it is verified whether the ideas are attractive from an economic and business point of view. At each decision “gate”, the team wants to get results from the completed activities and fulfilled criteria (Grönlund et al. , 2010). The initial steps involve the generation of ideas (brainstorming) and the identification of business opportunities, and the later steps are for development, testing, validation, and commercialization, as shown in Table 2. Table 2. Stage-Gate model (adapted from Cooper, 1990) Step 0: Generating ideas Generation of ideas from brainstorming, customers, complaints, R&D, competition, other markets, etc. Gate 1 Evaluation of ideas according to strategic positioning, project feasibility, technical feasibility, magnitude of opportunity, competitive advantage, synergy with the company's main businesses and resources and market attractiveness. Step 1: Preliminary assessment -- Preliminary investigation where size, potential and likely market acceptance are assessed; -- Rapid internal assessment of the product proposal (existing technology, patents, environmental and food safety issues); -- Collection of technical and market information, possible costs, and execution deadlines. Gate 2 Project is re-evaluated with more precise criteria than gate 1, and the decision to move on to step 2 is carried out. Step 2: Detailed evaluation -- At this stage the project must be clearly defined; -- Assessment of consumer needs and desires in order to define the product to be developed; -- Quantification of costs, product requirements and its market potential as well as project execution capacity (technologies and patents); -- Carrying out the concept test, where the likely acceptance by the customer of the new product is determined (perceiving consumer opinions and attitudes); -- Realization of the business plan: marketing plan (price, product, communication, distribution); economic-financial analysis; analysis of production and investment costs; definition of suppliers and ingredients and the distribution channel. Gate 3 Review of each of the activities in Step 2, verifying that they were carried out and the results were positive. Stage 3: Development -- Obtaining the product prototype (laboratory work for product development); -- Product evaluation with consumers. Gate 4 The development process is verified, ensuring that the work has been completed properly. Step 4: Testing and validation -- Project feasibility test: the product itself; the production process; customer acceptance, and; the economic overview of the project; -- Conducting product tests to verify quality and performance as well as assess customer reactions; accurately determine production costs; review the financial analysis (anticipated revenues and marketing and production costs). Gate 5 Last chance for the project not to move forward. At this gate it is important to review the financial plans (marketing and production costs) again. Step 5: Production and launch The marketing and production plans are carried out, with a monitorization of the product launch. Conclusions are drawn, weighing the positives and negatives of the launch. 8 2.4. Sensory Analysis Sensory analysis proves to be an essential tool that goes hand in hand with product development as a means of better perceiving the potential of a given product in the generalized view of the consumer, as well as seeing which innovation points are truly necessary or irrelevant for the quality of the indevelopment product. The use of sensory analysis is traced in history to wartime efforts of providing acceptable food to American forces and the development of the triangle test in Scandinavia, with a vital leap in the development of these methods being made by the Food Science Department at the University of California in 1965 (Meilgaard, Carr et al. 2016). Sensory analysis usage as a formalized, structured and codified methodology only came recently however, and new techniques and methods keep being developed today. Sensory testing evaluates alternative courses to select the one that optimizes value for money, and the principal uses of sensory techniques are in quality control, product development, and research. The primary function of sensory testing is to conduct valid and reliable tests that provide data on which sound decisions can be made. These methods hinge very heavily on human subjects as instruments which may prove at times difficult when the panellists are not trained in the perception required by sensory analysis (Meilgaard, Carr et al. 2016). Sensory analysis tests can be divided into three main groups: discriminative or differentiation tests, affective tests, and descriptive tests. Differentiation tests are based on the perception that the taster has of the difference between products and the objective is to detect these differences, whether they are global differences or specific attributes, but does not indicate the extent of this difference (Golob, Jamnik et al ., 2005). These tests include for example the duo-trio test and the triangle test. Descriptive tests involve the identification and description of the different sensory attributes of a given product, by a panel of properly trained panellists (or consumers in the most recent descriptive techniques), based on the use of quantitative response scales (Murray, Delahunty et al ., 2001). The main descriptive tests are the simple descriptive test, sensory profile test, and free-range sensory profile test. Finally, affective tests are tests in which the consumer is asked to express his preference or acceptance for a given product, and are used in new product optimization, definition of the new product's lifespan and analysis of the potential of the new product in terms of market (comparison with competitors). These tests are the most relevant to this study and are divided into: • Qualitative: focus group, focus panel, interviews; • Quantitative (the preference or acceptance by the current or potential consumer of an existing or under development product is evaluated). 9 A part of quantitative tests are three main tests, the preference test (in which the consumer is asked to indicate the product they prefer in relation to other(s)), the acceptance test (in which the consumer is asked to indicate the degree of acceptance of one or more products) and the acceptance/expectation test (in which the consumer is asked to indicate the degree of acceptance of various products and then assess the expectation they have of the pleasantness of these products through the packaging and other elements). Preference tests are tests that only compare or rank samples among themselves, not assessing the degree of acceptance on a scale. When a sample is chosen as preferred, it does not mean that it is positively appreciated by the taster, they can all be bad but this one is the least bad. In turn, preference tests are divided into hedonic paired comparison test, hedonic multiple comparison test and hedonic ranking test. The acceptance test on the other hand is a test used to assess the degree of acceptance of one or more products by consumers in which the coded samples are presented to the taster separately (monadic form) and in a balanced order between panellists (Lawless, Heymann, 2010). The panellists then score the agreeableness of each sample on a hedonic scale (the most common is the 9-point bipolar structured discrete scale). This test provides an independent evaluation of each sample, in which the acceptance of the products can be compared with each other. 2.5. Brewing Industry A specific sector of the drinks industry which also makes use of sensory analysis is the beer brewing industry (responsible for the production of beer). It can be said that it plays a significant role in Portugal’s economy, ranking 15th among the other sectors with the highest contribution to the economy. This sector is in rapid expansion with an increase of 7 to 120 breweries from 2011 to 2017, with most being microbreweries, which are responsible for a significant disruption in the beer market with the resurgence of craft beer. In the same time period, microbreweries have increased to 115 from only 1, which amounts to 95.8 % of the breweries in operation (Machado, 2019). Beer is consumed all over the world with a global production of 182 000 ML in 2020, which saw a decrease from previous years. In 2013, 197 000 ML of beer were produced, representing an increase of 34 % when compared to the year of 1998 (Conway, 2021a). When it comes to the leaders in beer production, China is highlighted as the number one producer of beer, with 34 111 ML as opposed to the 21 117 ML from the United States (Conway, 2021b). Data from Portugal is only available from 2019, where 710 ML of beer were produced, which is a significantly lower amount when compared to leading countries in beer production. When compared to previous years, a 10 growing trend is observable since 2016, the year that marked a significant reduction of 15.7 % of the beer produced in 2011 (Conway, 2021c). In 2020, the Belgian company AB InBev was the world's leading brewing group based on a production volume of about 46 740 ML. The company produced more than double the amount accounted for by the Dutch company, Heineken, which ranked second that year (Conway, 2021d). In terms of its consumption, beer is the second most popular beverage on a global scale, only behind spirits, and ahead of wine (WHO, 2018). In Portugal, the beer consumption per capita was 51 L in 2017, which represents a low portion of the consumption in Europe (average consumption in Europe is 71 L per capita ), falling short of countries such as the Czech Republic, Austria and Germany, which are responsible for a consumption of 138 L, 105 L and 101 L per capita , respectively (TBE, 2018). 2.6. Beer as a Product As a product, beer has been around for millennia, even if the processes behind its production were not fully understood. Beer is an ensemble of three biochemical processes, the formation of enzymes on the germinating grain, the conversion of starch to sugars by these enzymes, and the fermentation of the sugars by yeasts. Its origin cannot be fully determined; however, the oldest mention of beer dates to 2800 BC in Mesopotamia, with a description of the distribution of beer and bread to a workforce. This drink was very early noticed as free of dangerous germs, which meant that water could be purified through the fermentation process. So, for many centuries, beer replaced water as the common everyday thirstquencher (Kunze, 2004). However, the birth of a more official brewing industry came about in breweries of Christian religious foundations such as monasteries and nunneries, with beer being produced not only for personal consumption, but also as a form of payment. Its production process suffered changes throughout history and still continues to evolve today, and water, malt and hops as the only constituents of beer were defined in 1516 by the Bavarian purity law which regularized beer production from a legal standpoint, making it the first consumer protection law in the world (Kunze, 2004). Meanwhile, knowledge advanced and thanks to Louis Pasteur and his attribution of fermentation to microorganisms, new insights were now known on the fermentation of beer and the essential requirements to make it a stable product. In 1883 Emil Christian Hansen developed the single yeast propagation method (Hough, Briggs et al ., 1982) which was later further developed by Paul Lindner in 1893 with the drop culture method which allowed the use of pure yeast strains, diminishing contaminations and making possible the spread of light-coloured beers. Around this time marks an intensive phase of breweries being founded, with some still existing today as giant producers such as the Carlsberg breweries. During this time, other breweries 11 were expanding considerably with the largest producer being the Bass Brewery with an annual production of 250 000 L of beer. In the second half of the 19th century the brewing trade evolved on a global level which allowed the diffusion of knowledge on the science of brewing beer, with the creation of specialized journals, research laboratories and institutes, and brewing associations. Expert analysts also helped standardize knowledge with standard analysis methods. With this boom in the brewing industry the Bavarian purity law began to be abandoned by several countries, with the use of additives becoming increasingly popular (Kunze, 2004). 2.6.1. Raw materials Beer is a product of four raw materials, barley, hops, water and yeast, with adjuncts being sometimes used. However, these four raw materials are the ones that influence the final quality of the product the most, hence, knowledge of their properties and effects on the process and final product are crucial (Kunze, 2004). 2.6.1.1. Barley Barley is the main component of beer and is the source of starch required for conversion into a fermentable extract, so, a production of extract rich malts is necessary (by means of cultivation of suitable varieties). Barley is a cereal and is subdivided into two types and many varieties (Briggs, 1978), some of which may not prove very useful for beer production. These two types of barley differentiate on time of plantation, as winter barley is sown around September and spring barley around March and April. Winter and spring barleys can be subdivided in two varieties, two-row or six-row, which differ from one another on the arrangement of the corns on the ear axis (Briggs, 1978). Two-row barleys are preferably grown as spring barleys, where they possess all desirable features for malt and beer production. These barleys have larger amounts of useful contents and less husk, which translates to less polyphenolic and bitter substances. However, due to the need of this sector, the use of only one type of barley is impractical (Kunze, 2004), with two-row spring and winter, and six-row spring and winter barleys being used in the industry. Within these subclasses, barley still varies a lot in a number of different properties, with new varieties being worked on at all times, in order to improve brewing quality. The moisture content on the grains of barley is usually 14 % to 15 % but can vary from 12 % on dry harvesting conditions, to 20 % in wet conditions, with the latter having to be dried up for storage. As for the composition of the dry weight, it consists of 70.0 % to 85.0 % of total carbohydrates, 10.5 % to 11.5 % of protein, 2.0 % to 4.0 % of inorganic matter, 1.5 % to 2.0 % of fat, with the remaining 1.0 % to 2.0 % 12 being attributed to other substances (Kunze, 2004). The carbohydrates present are starch, sugars, cellulose, hemicellulose and gums, and these are the most important components of barley. The starch forms 50 % to 65 % of barley and is formed by condensation of glucose, representing a form of energy reserve needed for the initial growth phase of the seedling. It is composed by two different structures, amylose and amylopectin which are glucose residues with very different structures that influence the way they are broken down in malting and mashing (Asare et al. , 2011). Cellulose is present on low amounts, roughly 5 % to 6 %, in the husk, where it acts as a structural component. This carbohydrate has 1,4-linked β-glucose residues, meaning that it is insoluble and cannot be broken down by malt enzymes, having no impact on the quality of the beer. Hemicellulose on the other hand do and are composed of β-glucans and pentosans. The β-glucan consist of long chains of glucose with 1,3 and 1,4 bonds and is present at about 4 % in barley. Its molecules, when in solution, form micelles as a result of formation of hydrogen bonds, so, the breakdown of these micelles constitutes a great marker of quality on beer production, and it can occur in malting or in the mashing. Pentosans consist of chains of 1,4-D-xylose residues with arabinose residues linked to it and surround β-glucan in barley having to be broken down in germination even if their impact to quality of beer is insignificant when compared to β-glucan (Kunze, 2004). In regards of the nitrogen compounds, their content in barley can vary from 8 % to 16 %. The amount of protein in the finished beer is considered to be relatively small, with only a third of the proteins present in barley passing through the whole process. However, they still heavily influence the quality of the finished product, where they can form hazes. The proteins contained in barley are as follows: Glutelin (30 % of barley protein), Prolamin (37 % of barley protein), Globulin (15 % of barley protein, responsible for the formation of haze in beer) and Albumin (11 % of barley protein, completely precipitates when boiled) (Kunze, 2004). Since the wort is boiled, intact proteins cannot pass into the finished product, however their breakdown products can. These breakdown products are soluble in water and do not precipitate in boiling and the proportion of protein to protein breakdown products decreases during malting and brewing. These products are subdivided in high molecular weight (proteases and complex peptones which improve head retention of beer, but are also responsible for the formation of haze) and low molecular weight (amino acids and peptides) (Køie, Ingversen, Andersen, Doll, & Eggum, 1976; Klose, 2010). The amino acids are important for the yeast as they are used for the production of new cell substances (Kunze, 2004). Lipids are also a part of the composition of barley, even if at a low percentage (2 %), and are mostly present as fatty acids. Of these, the unsaturated fatty acids take a particularly high importance, as they are known to have beneficial effects on health, but most importantly, are needed for the structure of new 13 yeast cells wall. Some derivatives of these fatty acids are also considered responsible for ageing processes in beer after filling, so changes in the composition of beer in fatty acids is also important (Kunze, 2004). Barley also has in its composition some mineral compounds of importance, such as phosphates, without which fermentation would not be possible. Other substances present are polyphenols and tannins, which confer harshness and bitterness (and other adverse effects such as causing haze) to the final product when present at high quantities, vitamins, which are essential for a number of metabolic processes, and finally, enzymes, one of the most important components in the process of brewing beer. These enzymes are mostly formed during germination of the barley and are necessary for the breakdown processes that occur during mashing (Willaert, 2007). 2.6.1.2. Hops Hop refers to a perennial dioecious climbing plant of the hemp family (Moir, 2000). For brewing, only the inflorescences of the female plant are used, as these contain the bitter resins and essential oils responsible for supplying bitterness and aroma components to the beer, respectively. This plant has particular growth conditions, only being planted in special growing regions such as Germany, USA, Czech Republic and China (Kunze, 2004). After its harvest, the hops are dried (picked hops contain 75 % to 80 % water) and processed, usually into extracts or pellets, as to avoid quality reduction. Pellets are formed through the compression of dried hop milled powder and they subdivide in three types, pellets type 90, enriched pellets and isomerized pellets, each with different effects on beer quality (Bamforth, 2006). The composition of hops is vital for the quality of the produced beer, consisting of 18.5 % bitter substances, 0.5 % hop oil, 3.5 % polyphenols, 20.0 % protein and 8.0 % minerals (Kunze, 2004). Of these, the ones that attain more importance are the bitter substances and hop oil. These bitter substances, or hop resins are initially present in the hop plant as β-acids that are only slightly bitter; however, in the course of the maturing process, these are converted into their respective α-acids which are considerably more bitter (Likens et al., 1978). This is one of the factors that affects the growing conditions for desirable hops for brewing, as the conversion to α-acids only occurs at cooler temperatures and damp environments. As the type of α-acids present in the hop heavily influences the quality of the bitterness in beer, only certain varieties of hops are selected for growing. An important aspect of the bittering in beer is the isomerization of the α-acids cohumulone, humulone and adhumulone, that occurs during wort boiling according to Figure 2, and produces the respective soluble iso-α-acids (Ocvirk & Košir, 2020) which find their way to the finished beer. 20 p /bar; p/ (100 kPa) Figure 3. Forced carbonation chart for beer, relating temperature ( T ) with the pressure ( p ) (adapted from O’Leary, 2008). Blue-under carbonated; Gray-lightly carbonated (British ales, stouts and porters); Greenmoderately carbonated (lagers, ales, most beers); Yellow-highly carbonated (lambics and wheat beers); red-over carbonated (certain types of ales). The HACCP relates the quality of raw materials, processes, product composition, storage conditions, type of packaging, among others, based on seven principles (Baptista, Pinheiro, & Alves, 2003; Mortimore & Wallace, 2013), according to the Codex Alimentarius: 1. Carry out a risk analysis; 2. Determine critical control points (CCPs); 3. Establish critical threshold(s); 4. Establish a system to monitor the control of CCPs; 5. Establish corrective action to be taken when monitoring indicates that a specific CCP is not under control; 6. Establish verification procedures to confirm that the HACCP system works effectively; 7. Establish documentation regarding all procedures and records appropriate to these principles and their application. In Portugal, it was implemented with the decree 67/89 of March 18th, which establishes the general rules for the hygiene of foodstuffs, as well as the modalities for verifying compliance with these rules, which was later amended by decree 425/99 of October 21st, 1999. This decree was revoked in decree 113/2006, however, the enforcement of HACCP practices is present in EC Regulation no. 852/2004. T /ºC 21 The ISO 9001 on another hand is a quality management system and deals only with the quality of a product, defining activities for which the brewery must provide appropriate control, but not how these aspects must be controlled (Tarí, Molina-Azorín, & Heras, 2012). This Standard is also present in most breweries nowadays, complementing the activity of other systems in place such as the HACCP to offer quality and security to the consumer. Quality examinations of beer are usually in order, as to assure consumers of its quality and safety, and can be done by beer tasting, microbiological monitoring, and chemical and physical properties examination. Through beer tasting, many substances can be perceived, as well as properties of the finished product such as the amount and stability of the foam, colour, and turbidity (Parker, 2012). Quality determining parameters such as taste and aroma however are more difficult to measure, and in order to draw specific conclusions, a specific and strict method must be put in place. These analyses fall under the category of sensory evaluation, and include dilution tests, sweetness comparison tests, bitterness comparison tests, duo-trio tests and triangular tests. Usually, the sensory evaluation performed in breweries is done among the available workforce, which with experience make increasingly more correct distinctions and conclusions. Other tests are also applied, among the consumer as to get their perspective on the product, namely, preference and acceptance tests (Costell, 2002). In the case of consumers, descriptive tests could also be applied, at the risk of losing reliability of results, as hundreds of descriptors can be used for beer. The microbiological examination is another form of quality control, as it is possible for microorganisms that cause spoilage to pass from the production line to the finished product (Kunze, 2004). If not detected in early stages these can make beer turbid and produce substances through their metabolic pathways that spoil its taste. In regard to microorganisms that could be found in beer, three categories exist, the indirect beer spoilage organisms (harmless accompanying microorganisms), the potentially damaging microorganisms, and the obligatory spoilage microorganisms. The first usually cannot grow in beer and consequently die, serving however as indicators to the presence of undesirable microorganisms. The potentially damaging ( Lactobacillus casei , Lactococcus lactis and Enterobacteriacea) are the microorganisms that can grow on beer given that certain conditions are met, such as oxygen content, low pH and bitterness, which makes these attributes important factors to control (Schneiderbanger, Grammer, Jacob, & Hutzler, 2018). The obligatory microorganisms in beer ( Pectinatus cerevisiiphilus and Megasphaera cerevisiae ) are singled out due to their particularly damaging effect and favourable conditions for growth in beer (Kunze, 2004). Frequent checks are preferable in breweries at several segments of the production line as a means to determine where and how microorganisms enter the 22 brewing line, and to eliminate them accordingly, with countermeasures being introduced. However, at smaller breweries, beer quality can be difficult to ensure through microbiological monitorization, and so, physical and chemical properties examinations attain a higher prevalence. These examinations focus on a variety of parameters and are done along several steps of beer production (Bamforth, 2006). They include analysis of exctract concentration, determination of pH, diacetyl content, bitterness units, CO2 content, colloidal stability and other measurements. Determination of extract concentration and pH along processes such as mashing, boiling and fermentation also help keep these values in check for the specific style of beer being brewed (Habschied, Mastanjević, 2021). 2.8. Beer types Beer can be classified according to many factors, the main being type of fermentation, with top fermenting beers and bottom fermenting beers (Capece et al. , 2018). Top fermenting beer yeasts develop larger amounts of fermentation by-products, therefore conferring a more complex aromatic profile to the beer. The styles of beer included in this category are wheat beers, Berliner Weisse, Altbier, Kölsch, Ale, Stout, Porter, Trappist and Lambic (Bamforth, 2006). On the other hand, bottom fermenting yeasts confer a cleaner profile to the fermented product, and styles of beer included in this category are the Pilsner, Lager, Bockbier and Doppelbock beers (Bamforth, 2006). 2.8.1. Grape Ales and Lagers Ale beers are produced usually with well modified malts and adjuncts, which in conjunction with the complex aromatic profile conferred by the yeast give a more complex final product. The alcohol content, by volume ( ABV – alcohol by volume), of these beers can range from 3 % to 10 % and the bitterness varies greatly, with Indian Pale Ale being very heavily hopped, and mild ales being sweeter (Bamforth, 2006). Lager beers account for 80 % to 90 % of bottom fermenting beers and are characterized by an ABV between 4.7 % and 5.3 % and a moderate bitterness ( IBU from 18 to 23) (Bamforth, 2006). The Grape Ale and Lager are therefore derivatives of these styles that involve an addition of grapes or grape must. The most notorious beer style that involves the addition of grape must is the IGA, Italian grape ale (Garavaglia, 2018), defined as a communion between beer and wine (Garavaglia, 2020), whose pioneer, Nicola Perra, used sapa , grape must syrup obtained by long boiling. Since then, the main raw materials were conventionally defined as pils or pale malt, with possibly some adjuncts or special malts. The grape must used (pasteurized or not) can be introduced at different stages and different quantities with the final product varying highly. This addition can occur at boil, primary/secondary fermentation, or 23 aging, with quantities ranging from 5 % to 10 % of the final volume, although, they can go up to quantities such as 40 %, all depending on the creative decision of the author of the beer, since despite existing a norm for this style, no set of rules is actually defined, making this style highly experimental and everevolving (Marin et al ., 2021). Also, a factor of this variety in the style is the yeast used and therefore fermentation profile, which can be neutral or fruity. A wide range of hops can also be used, albeit at low amounts as not to give the final product an excessive beer character. Another interesting aspect of these beer styles is their interconnection with sour beers, as often grape ales could be soured, due to the already present acidic element of the grape must. As for standard quality parameters, the original extract ( ºPlato ) is usually between 10.7 and 22.5 with a final extract of 1.7 to 3.7, bitterness ( IBU ) between 10 and 30, colour ( EBC ) between 10 and 60, and ABV ranging from 4.8 % to 10 % (de Francesco, Marconi et al . 2021). The wide range of the different parameters indicates the complexity of this style (Alfeo, Todaro et al ., 2019). This is a style complicated to define as various materials and various characteristics interplay, being therefore a style very subject to innovation and change, where multiple of the above-defined parameters can be changed for a significantly different final product (de Francesco, Marconi et al . 2021). Given the acidic character that the grape must confers, beers produced with this component are sometimes paired with souring techniques in order to produce complex sour beers. The sour beer is known as a beer with intentionally sour taste where, traditionally, the acidic character is the result of spontaneous, mixed fermentation, implying a variety of bacteria and yeast species participation. A very popular style that is a result of this more traditional souring is the lambic beer. The traditional fermentation used to produce them does not apply pure yeast cultures but relies on spontaneous, environmental inoculation. The fermentation and maturation process of water, barley malt, unmalted wheat, and aged dry hops is carried out in horizontal oak or chestnut barrels (de Roos & de Vuyst, 2022) and can take up to three years, resulting in a sour, fruity beer with a complex microbial consortium and flavour profile that not only can be drunk as such but also serves as the basic product for other, lambic-derived beers (Bongaerts et al. , 2021). It is characterized by different microbial species belonging to the enterobacteria, acetic acid bacteria, lactic acid bacteria, and yeasts (de Roos & de Vuyst, 2019). However, the difficulty in maintaining a standardized quality due to the spontaneous aspect of the fermentation, paired with the time it takes to finish production gave rise to a different souring technique, an addition of pure acidifying organisms chosen with the desired characteristics in mind. These organisms, such as nonSaccharomyces yeasts ( e.g. Brettanomyces ) or acidifying bacteria ( e.g. lactic acid bacteria, acetic acid bacteria) can be used before (pre-alcoholic fermentation) or after (secondary fermentation) the main 24 fermentation with brewer’s yeast, resulting in differing final products. Acidification through pre-alcoholic fermentation with lactic acid bacteria is a known method used nowadays, which contributes to the sensory profile of the beer not only through the production of lactic acid, but also regarding volatile compounds and organic acids (Dysvik et al ., 2019). 25 3. Materials and Methods With the goal of producing a grape-based beer three main product tests were conducted which led to a final test solely made from the conclusions drawn from the previous three. The three main product tests served as a basis to mainly test three different styles of beer through which a final one was selected. Also, several other factors were tested, here considered secondary aspects, such as malts, hops, and techniques used ( e.g. kettle souring and dry hopping). These secondary aspects were not kept isolated from test to test like the primary aspect, the style of beer, meaning that knowledge gained on the first product test, the Grape Ale, was used to better develop these aspects on the second product test, the Grape Lager, and then again for the third, the Grape Weiss. Finalized these three main tests, both the secondary aspects refined throughout the product tests, and the primary aspect, the beer style, were imparted to the final product test, as seen in Figure 4. Here the work carried out in this study is summarized, the dashed lines implying the improvement of the secondary aspects along the course of the study, and the arrows meaning a possibility for a chosen beer style, which was decided after production of the Grape Ale, Grape Lager and Grape Weiss. FIGURE 4. ORGANIZATION OF THE STUDY. First Product Test Third Product Test Second Product Test Final Product Test Grape Ale Grape Lager Grape Weiss 26 3.1. Brewing and fermentation equipment An equipment that simulates the process of the brewhouse was used for brewing, the Grainfather, which is an all-in-one brewing system that allows the use of crushed grain, and mash, sparge and cool within the same unit. It is therefore an equipment fit for experimenting and innovating. Built into the boiler is a temperature control, heating elements, recirculating pump and counter flow chiller. The Grainfather is made of materials that grant the quality required for the brewing process such as 304 grade stainless steel, tempered glass, magnetic drive pump, copper cooling coil, etc. It has a 30 L capacity, meaning it can produce approximately 23 L of craft beer at one time, being also dotted with a magnetic drive pump operating at a rotational speed of 1800 min-1, that helps extract all sugars in the grain giving optimal brewing efficiency. After boiling, the wort is cooled through the use of the aforementioned counter flow chiller directly onto a separate fermenter. Here, temperature can be measured, along with visual verification of fermentation. Additionally, samples were taken through the use of a hose, allowing for sensory evaluation (tasting) and measurements of pH and extract concentration, as ºPlato (where a ºPlato of 10 corresponds to an extract concentration of 100 g/kg) during the production and fermentation process. Evaluation of pH and extract concentrations were also made during fermentation, and for both brewing and fermentation, samples were collected, cooled for a time to room temperature and analysed with pH-meter Model 20 (Crison) for pH and Refractometer PAL-1 (Atago) for extract concentration where values are obtained in Brix , which were later converted to ºPlato . Measures of CO2, O2 and ABV (alcohol by volume) were also made of the finished product, with a CboxQC AT-line and filling device PFD (Anton Paar) for CO2 and O2, and an alcohol analysing system, Alcolyzer (Anton Paar), for ABV . 3.2. Raw Materials 3.2.1. Malts Three different types of malted products were used depending on the beer type, Finest Lager (from Simpsons Malt), Cara Aroma (from Weyerman) and Wheat Malt (from Simpsons Malt). Technical specifications for malted and unmalted cereals used are present in Appendix A, Table A1, and amounts used in the different product tests are in Table 3. 27 3.2.2. Hops In terms of hops, Citra hops (from Yakima Chief Hops) and Tettnang Tettnanger (from Charles Faram) were used. Technical specifications for hops used are present in Appendix A, Table A2. Table 3 presents the amounts used for the different product tests. 3.2.3. Yeasts and Bacteria Yeasts were again used accordingly with the different beer types (Table 3), and the main focus for this study were the SafAleTM US-05 (Fermentis), SafLagerTM W-34/70 (Fermentis), SafAleTM T-58 (Fermentis). Other than these, an acidifying bacterium was used, WildbrewTM Helveticus pitch (Lallemand), and a yeast typically found in wine production, Lalvin QA23TM (Lallemand). 3.2.4. Adjuncts Among the adjuncts vital for these product tests (Table 3) were the grape must from the grape noble variety of Loureiro courtesy of Esporão’s Quinta do Ameal, Flaked Oats (from Simpsons Malt), Flaked Wheat (from Simpsons Malt), Go-Ferm ProtectTM (Lallemand), DAP, or diammonium phosphate, Servomyces (Lallemand), and CaCO3 and Citric acid (Sameca). 3.2.5. Salts Salts used included only CaCl2 and CaSO4 (Sameca) with amounts specified in Table 3. 3.3. Production Process The four product tests followed the same procedure with small differences. For beer production with the Grainfather equipment the malt was weighed and milled, in anticipation to the mashing, and added to the brewing equipment alongside water from a reverse osmosis system (volume of water is specified in Table 3). Mashing followed this first step, with different temperature ranges for the different product tests according to the mashing guidelines in Table 3, and salts were added. The final product test followed the same mashing guidelines as the Grape Lager. Grape must was added in this step for the Grape Ale only, in this case half of the total amount of must used, 5 L. With the brewing equipment used, recirculation of the wort occurred during the whole process of mashing, at the end of which the wort was filtered. After filtration, boiling procedures take place, at 100 ºC for 60 min for the first three products, and 90 min for the final product test. With the grape ale only, a first boiling occurred before kettle souring, for 15 min, with an addition of lactic acid bacterium ( Lactobacillus helveticus) after the wort cooled to 43 ºC. The 28 souring step ended when the wort hit the target pH of 3.39. Grape must was mainly added in this step, only 5 L for the Grape ale, and 10 L for the rest, all at 15 min remaining in the boiling and represented 43.5 % of the mashing volume. Hops were also added in this step, with 20 min remaining for the Grape Ale, and 60 min for the other three product tests. The sterilized wort then proceeded to cooling with the counter flow chiller and sent into a fermentation vessel, where the yeast was added in a uniform manner through direct pitching. For the Grape Weiss and final product test, an additional use of yeast was performed to restart fermentation. The contents were moved to refrigeration at the end of fermentation and dry hop additions were made for the Grape Ale and final product test (its second dry hop addition). For this final product test, a cleaning of H2S aroma was necessary and carried out before refrigeration by passing the maturing beer through a copper surface (as well as a first dry hop addition). Maturation occurred at 7 ºC for 15 days for the Grape Ale and Grape Weiss, 20 days for the Grape lager and 30 days for the final product test. Finally, after priming with CO2, bottling procedures took place. During fermentation and maturation, samples were taken when possible for the measurement of temperature, ºPlato and pH. Specifications for the amounts of raw materials used in each product test are represented in Table 3. The represented dosages of yeasts and adjuncts (percentage of grape must, DAP, Servomyces and Go-ferm) are the planned concentrations for each product test and not the exact values as data on the final volume of the wort both in mashing and boiling is unavailable. 3.4. Sensory Analysis Sensory analysis trials used in order to better perceive the consumer’s point of view on the products fabricated was an acceptance test, where a grading of 1-5 on a hedonic scale was requested of 15 panellists, 11 men and 4 women, from the ages of 22 to 58 on the categories of appearance, aroma, bitterness, flavour, palatefulness and global appreciation, and an answer of “Yes/No” on the category of intent to purchase according to the evaluation sheet designed, adapted from an existing Sovina’s evaluation sheet (Figure 5). The panellists ranged from people with experience on the beer industry and therefore more trained to understand subtle differences in the product, to others who were not trained. Also, an important factor in these sensory analysis assays were the varied preferences in the panellists and their like/dislike of beer as a product in general. They were implemented 2 h before lunch and crackers and water were served so that the palate could be cleansed in between the different beers. For the first sensory evaluation, the three first product tests were analysed, and the beers were served according to the same order to all panellists, going as follows: Grape Ale, Grape Lager and lastly, Grape Weiss. Characteristics for these products were not given on an initial phase to recreate a blind test and 29 panellists only had to grade the appearance, aroma, bitterness and palatefulness, filling the flavour category with a descriptive term of their choice. Table 3. Raw materials and process guidelines used for the product tests Grape Ale Grape Lager Grape Weiss Final Product Test Salts CaCl2 2 g CaCl2 2 g CaCl2 2 g CaCl2 2 g CaSO4 1 g CaSO4 1 g CaSO4 1 g CaSO4 1 g Malts Finest Lager 2.0 kg Finest Lager 3.5 kg Finest Lager 2.0 kg Finest Lager 7.0 kg Cara aroma 0.2 kg Flaked Oats 0.7 kg Wheat Malt 2.0 kg Wheat Flakes 1.0 kg Hops Citra 20 g Citra 13 g Tettnanger 20 g Citra 15 g Citra (dry hopping) 10 g Citra (dry hopping) 10 g Citra (dry hopping) 10 g Yeast/Bacteria US-05 11.5 g (52.2 g/hL) W-34/70 11.5 g (52.2 g/hL) T-58 23.0 g (104 g/hL) W-34/70 23.0 g (104 g/hL) Helveticus pitch 2.00 g (10.0 g/hL) QA23 11.0 g (50.0 g/hL) Adjuncts Grape Must 5 L (in mashing) Grape Must 10 L Grape Must 10 L Grape Must 10 L 5 L (in boiling) GoFerm 2.85 g (12.9 g/hL) GoFerm 3.60 g (16.4 g/hL) Servomyces 1.00 g (4.54 g/hL) Servomyces 1.00 g (4.54 g/hL) DAP 2.85 g (12.9 g/hL) DAP 3.00 g (13.6 g/hL) DAP 3.00 g (13.6 g/hL) DAP 3.00 g (13.6 g/hL) Water Mashing 12.0 L 20.0 L 8.0 L 28.0 L Sparging 3.0 L 5.0 L 6.5 L -- CO2 2.6 g/L 2.6 g/L 2.6 g/L 2.6 g/L Mashing guidelines 64 ºC 60 min 64 ºC 30 min 55 ºC 15 min 64 ºC 30 min 78 ºC 15 min 68 ºC 30 min 64 ºC 45 min 68 ºC 30 min 75 ºC 10 min 76 ºC 15 min 75 ºC 10 min Boiling Guidelines 100 ºC 60 min 100 ºC 60 min 100 ºC 60 min 100 ºC 90 min On a second phase, the characteristics of the beer were given to the panellists, and a completion of the grading (flavour, global appreciation and intent of purchase) was achieved. The second sensory evaluation trial was conducted at a later date and only the final product test was evaluated. 36 Figure 7. Global appreciation and intent of purchase of the Grape Ale beer based on number of answers ( n ). Out of the characteristics evaluated in Figure 8 there were some positive conclusions, with the appearance and aroma of the beer being positively assessed, with 46.6 % and 40.0 % of panellists, respectively, rating a 4 in the used preference scale. These results go accordingly with the planned objectives, and in the most part mirrored the preliminary tasting that occurred by the time of production. However, from the point of view of the consumer, it appears that a grape-based beer would beneficiate from a higher bitterness than the one utilized for this product test, which differs from the conclusions and plans drawn by the author of the study. Figure 8. Sensory analysis regarding appearance, aroma, bitterness, flavour and palatefulness of the Grape Ale beer based on number of answers ( n ). 0 2 4 6 8 10 12 12345 n Appreciation (1-5) 0 2 4 6 8 10 12 Yes No n Intent of Purchase 0 1 2 3 4 5 6 7 8 12345 n Appreciation (1-5) Appearance Aroma Bitterness Flavour Palatefulness 37 4.1.2. Second Product Test – Grape Lager The following step was testing a bottom-fermented beer, which in light of the metabolism of the picked yeast, W-34/70, should produce a cleaner and more neutral beer, on where an addition of the same amount of grape wort as the previous product test should not go unnoticed. The chosen yeast is a lager yeast known for producing neutral beers with a good balance of floral and fruity aromas, that gives clean flavours and highly drinkable beers. It presents a low production of esters and higher alcohols with an apparent attenuation of 80 % to 84 % if kept at a temperature range of 12 ºC to 18 ºC. W-34/70 yeast has a fast sedimentation time, an alcohol tolerance of 9 % to 11 % and recommended dosage is 80 g/hL to 120 g/hL (Fermentis 2022b). This yeast was added at a concentration of 52.3 g/L which could explain the slow start for the fermentation, and the fermenting product was kept at 12 ºC with the assistance of a cooler. In addition to the designated lager malt, an unmalted cereal was also used, flaked oats to add texture and mostly, to create a certain degree of turbidity that could be an interesting feature for a grape must related product. The Flaked Oats used are an unmalted product containing high levels of lipids, β-glucans and gums, which impart a silky mouthfeel and creaminess to beer. The more Flaked Oats in the grist, the greater the effect, but upon passing 20 % of the grist, the β-glucans and gums would have a slowing effect on wort run off (Simpsons Malt, 2022b). As for the mashing guidelines, the wort went through three stages in the ramp up of temperature, starting at 64 ºC for 30 min, then 68 ºC also for 30 min, and finally, a mash out at 75 ºC (10 min). These ramps were designated in order to maintain the sweet aftertaste felt in the first product test, as at 68 ºC α-amylase is at its optimal temperature activity, meaning that a higher conversion of starch into maltose, maltotriose and dextrin exists, the last being a non-fermentable sugar by the yeast. As seen with the first assay, a higher volume of water had to be used to enable mashing, which was then deducted in sparging. The grape must was added in its entirety (43.5 % of mashing volume) to the boiling wort, during the final 15 min of this stage, as to sterilize it and rid it of potential dangers to the finished product, but still maintain characteristics inherent to its volatile compounds. No dry hop was utilized for this product test to better check if this fermentation type suited the grape wort, and if its taste and aroma was noticeable on the finished product. However, the same type of hop was added during boiling since the citric aroma was enjoyable in the first product test (a low amount is used in order to have a product with low bitterness, since it could make a complex final product in conjunction with the acidity provided by the grape must). Despite the slow start fermentation went mostly as expected, as seen in Figure 9 (specific day data in appendix B, Table B2), but suffered some setbacks close to its ending, as it stopped at 6.8. This 38 corresponds to an apparent attenuation of 46.6 %, significantly lower than the supposed 80 % to 84 % this yeast is capable of. These results could be a consequence of the low dosage of yeast used, the 68 ºC mashing, or a lack of nutrients such as nitrogen for the yeast metabolism. Another explanation could be the presence of the grape wort, or even a high ethanol content. The W-34/70 yeast has an alcohol tolerance of 9 % to 11 %, and perhaps due to the prolonged storage of the grape must (at cold temperatures, which only slows down fermentation by wild strains of yeasts and bacteria) the ABV of this beer was 7.5 %, higher than expected even though fermentation did not convert all the fermentable sugars. Priming was achieved with the same conditions as the previous beer, as the refreshing aspect was something to maintain. The final product presented a gold colour, with an interesting turbidity, not common on a lager beer. As for its aroma, malt and grape must were noticeable, with also citric hints. The grape must was also slightly present on the flavour of the finished product, especially on the aftertaste. The citric element provided by the Citra hops was present at very low levels, and since it did not mask the grape must, a dry hopping could be employed in following product tests. Given that fermentation ended at a high ºPlato , the expected sweetness is also present which is not necessarily a negative point since it creates a pleasant balance together with the acidity from the grape must. Figure 9. Variation of temperature ( T ), extract concentration ( ºPlato ) and pH during fermentation time ( t ). The ABV of 7.5 % gives this product a fuller body and heavier mouthfeel which was not something expected, but since the conditions of the grape wort could not be fully controlled and it already underwent a certain degree of fermentation, it was an aspect that had to be present for the next product tests. Overall, 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 5 10 15 20 ºPlato ; pH T /ºC t /d T ºPlat o pH 39 the bottom fermentation proved to be very beneficial for the addition of grape wort, as a more neutral base made way for the grape must to be present in both the taste and aroma. This product performed better overall than the other tests in terms of the consumer’s opinion, which is highly reflected by the 53.3 % of panellists that corresponded to a 4 in the hedonic scale used for the global appreciation and the 80.0 % of panellists that voted “Yes” when inquired about intent to buy this product. An interesting result is the 20.0 % of panellists that graded 2 on global appreciation, but then again, this only reflects the fact that a grape-based product has a niche place on the market and is still very unknown to the public eye (Figure 10). As for the other categories appraised, most had very positive results, and the bitterness was the category where panellists were most divided (Figure 11), perhaps the most unexpected, as during production a low bitterness was found to be positive for this product, conclusions not shared by the panellists. As for flavour and aroma, perhaps the most significant of the appraised categories, the grape lager was highly ranked amongst the panellists, with a majority of answers grading 4 in the used scale. Figure 10. Global appreciation and intent of purchase of the Grape Lager beer based on number of answers ( n ). When asked for descriptors on this particular product, only one participant attributed vinic notes to the beer, which implies that on the larger consumer view that are unaware of the production process, it still is hard to detect the grape must. 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 n Appreciation (1-5) 0 2 4 6 8 10 12 14 Yes No n Intent of Purchase 40 Figure 11. Sensory analysis regarding appearance, aroma, bitterness, flavour and palatefulness of the Grape Lager beer based on number of answers ( n ). 4.1.3. Third Product Test – Grape Weiss The following fermentation type tested was, again, a top fermented product, but, that of a Weissbier, which is known for a very different aromatic profile from other beers, on account of the metabolism of the yeasts generally used for these beers, which can convert ferulic acid from the malt to 4-vinylguaiacol, responsible for a clove aroma and flavour (McMurrough, Madigan et al. 1996), and isoamyl alcohol to isoamyl acetate through the activity of an isoamyl alcohol acetyltransferase (Lilly, Lambrechts et al. 2000), the substance that gives beer banana flavour and aroma. Malts used included a lager malt and the fundamental wheat malt which meant that a mashing rest at 55 ºC was necessary for the activity of β-glucanases. Wheat malt (Simpsons Malt, 2022c) can be added at 5 % to most Light Beers to increase foam stability without affecting haze or flavour and used at up to 70 % as a base malt for Wheat Beer, it imparts a bready flavour and characteristic citrus notes. The mashing started at 55ºC for 15 min, with a following rest at 64 ºC for 45 min, and finally, a mash out at 76 ºC. This assay showed the least inconsistencies with water volume used for mashing and sparging, as the planned amount was barely enough (only an extra 2 L were added). Since Weissbiers are usually known for their phenolic and fruity ester components, citric hops were deemed unnecessary and a hop more commonly present amongst wheat beers was used, Tettnanger, albeit at still a low quantity, as a low bitterness was wanted. This hop has a significantly high farnesene content which gives it a soft spiciness and a subtle, balanced, floral and herbal aroma. It is also great as a dual-use hop (Hoplist, 2022b). Grape must was added at the same stage as in the previous product test and in the 0 1 2 3 4 5 6 7 8 1 2 3 4 5 n Appreciation (1-5) Appearance Aroma Bitterness Flavour Palatefulness 41 same amount. The yeast used, T-58, is a highly phenolic yeast generally used for its strong fermentation character, intense fruity and phenolic flavours, such as those of banana, clove and pepper. Suitable for wheat-base beers, this yeast produces a high quantity of esters and medium for higher alcohols as a result of its metabolism. It has an apparent attenuation of 72 % to 78 % if kept at the temperature range designated by the manufacturer of 18 ºC to 26 ºC. T-58 yeast also presents an alcohol tolerance of 9 % to 11 % and a medium sedimentation time. Recommended dosage is 50 g/hL to 80 g/hL (Fermentis, 2022c) and the planned concentration was 104 g/hL. This product test differs from the previous in the use of Servomyces, a naturally enriched biological yeast nutrient, substituting the Go-Ferm nutrient. The propagation and drying process of this brewing yeast has been specifically designed to accumulate a range of trace minerals and elements that are essential or limiting during alcoholic fermentation. This yeast nutrient decreases fermentation time significantly, improves yeast sedimentation, stimulates uptake of maltose and maltotriose (higher alcohol levels), stimulates protein synthesis and yeast growth (higher biomass production during propagation), and helps eliminate harsh sulphur notes to produce a smoother and more balanced beer (Lallemand Brewing, 2022b). As shown in Figure 12 (Table B3 in Appendix B), fermentation stopped once again early, at a ºPlato of 6.81, at which point 500 mL of T-58 yeast in high activity from another fermentation were inoculated to attempt a restart, however, the ºPlato only lowered to 6.62, which implies an apparent attenuation of 59.6 %. This result is still higher than the previous ones which could disprove the theory that the growing alcohol percentage of the grape must could be influencing negatively the fermentation process, however, other factors could have intervened, such as the lower original ºPlato , more ideal pH for the yeast, or bigger supply of nutrients present such as nitrogen availability from the malts used. Still, the attenuation achieved was lower than expected since the T-58 yeast has an apparent attenuation of 72 % to 78 %, and the ABV for this beer was 6.9 %. The finished product presented a pale gold colour with a cloudy turbidity common in wheat beers. The aroma was phenolic and slightly fruity, and the banana, or isoamyl acetate was more present on the flavour. Grape must was not quite traceable on either the aroma or the flavour, as the aromatic profile of this fermentation was considered not very suiting for the objective of this study. The strong phenolic and ester character provided from the yeast gave conflicting results as the clove and banana aroma conflicted with the grape must aroma. For the flavour, the same results were obtained. These results do not particularly imply a failed product as the resulting aroma and flavour was interesting, however, do not go 42 according to the objective of this study which was to see what fermentation style and other characteristics allowed for the addition of grape must to be well noted on the finished product. Figure 12. Variation of temperature ( T ), extract concentration ( ºPlato ) and pH during fermentation time ( t ). In terms of the sensory analysis trials results, this product test scored very similarly to the first product test, albeit to a more positive side. Appearance and aroma were the categories that were graded the best, with 60.0 % and 53.3 % of panellists to give a 4 out of 5 on the scale used, respectively as seen in Figure 13. Figure 13. Sensory analysis regarding appearance, aroma, bitterness, flavour and palatefulness of the Grape Weiss beer based on the number of answers ( n ). 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 ºPlato ; pH T /ºC t /d T ºPlato pH 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 n Appreciation (1-5) Appearance Aroma Bitterness Flavour Palatefulness 43 The aroma was found by the panellists to be pleasant with most attributing it a 4, which was not similar to the preliminary analysis conducted during production, as the phenolic and ester character was considered to conflict with the grape must. However, it is not very surprising that it ranked high with the panellist which evaluated the aroma during a blind test, where the clove and banana aroma seemed predominant and pleasant. Besides the aroma, bitterness was also perceived differently by the consumer and found to not be quite right. On the intention of purchase panellists were almost evenly divided with 46.6 % of “No” and 53.3 % of “Yes” (Figure 14). Figure 14. Global appreciation and intent of purchase of the Grape Weiss beer based on number of answers ( n ). 4.2. Culmination of the study -- Final Product Test According to the ideas that were gathered throughout this study and the production of the first three product tests, a final test was designed, following the characteristics that were considered successful from all the product tests. The main point, the fermentation style, was chosen from the second product test, which was the one where the addition of grape must was most noticeable. The W-34/70 yeast was used, at a concentration of 104 g/hL. Malts used were also meant to be kept from that product (amounts were increased for a higher final volume, but the rest of the conditions were adjusted so that the ABV could be similar) but flaked oats were not available and thus substituted with flaked wheat, which also confers turbidity and creaminess to the final product. Flaked Wheat is therefore an unmalted product that gives great foam stability and mouthfeel. This unmalted wheat product is packed with high molecular weight protein, which help give foam stability, as well as greater mouthfeel and turbidity, being traditionally used for head retention and to supplement wort nitrogen (Simpsons Malt, 2022d). The hop chosen was Citra, at a low quantity for a low bitterness, and two dry hopping additions were done, one during the highest activity of the QA23 yeast, high krausen, and the second before lowering the 0 2 4 6 8 10 12 12345 n Appreciation (1-5) 0 2 4 6 8 10 12 Yes No n Intent of Purchase 44 temperature of the product for maturation. The QA23 yeast was used in an attempt to restart fermentation, and it is a yeast that is typically used for wines where fruit expression is desired, as it has a high β-glucosidase activity, which helps cleave non-volatile aromatic compounds into their volatile state. As for its oenological properties, it has an average lag phase and vigorous fermentation, low relative nitrogen demand, very low H2S production, alcohol tolerance of 16 %, low SO2 production, fermentation temperature (14 ºC to 28 ºC), competitive killer factor present, malolactic-bacteria compatibility and low foam production (Lallemand Brewing, 2022c). Higher volumes of water had to be used for mashing, a common trend observed amongst these product tests, which implied no sparge water as the volume limits of the equipment were reached. Fermentation again stopped early, at ºPlato of 7.6 as seen in Figure 15 (and Table B4, appendix B), which corresponds to an attenuation of 53 %. In the case of this product, besides the other already stated reasons for the fermentation to stop such as the growing alcoholic percentage of the grape must, problems subsisted throughout the process with the temperature, as it was impossible to maintain at the supposed 12 ºC ideal for the yeast used (W-34/70). Instead, the temperature was unstable and varied between 13 ºC and 20 ºC which is not adequate to the yeast metabolism. Perhaps due to these reasons a sulphur-like aroma was detected nearly the end of the fermentation and measures had to be taken so that the finished product did not present it. For this, the product underwent copper treatment, and a removal of this offflavour was successful. Before this treatment, an attempt at restarting the fermentation occurred with the starter of wine yeast QA23 which has a higher alcohol tolerance than W-34/70, however, this attempt was unsuccessful. Figure 15. Variation of temperature ( T ), extract concentration ( ºPlato ) and pH during fermentation time ( t ). 0 2 4 6 8 10 12 14 16 0 5 10 15 20 25 0 2 4 6 8 10 12 14 ºPlato ; pH T /ºC t /d T ºPlato pH 45 The finished product presented an appearance alike to the second product test, as the raw materials in its basis are very similar. As for the aroma, as a result of the dry hop additions of the Citra hop there was a very pleasant citric aroma that complements the grape must aroma. The flavour however was where this product failed, as due to the problems that surfaced during fermentation a high alcoholic content could be detected, and a strong sweetness was present. The grape must was nowhere to be found in the flavour, as the predominant flavour was sugary and alcoholic. As a consequence of this, the mouthfeel was also off, and the product presented a very heavy body. Unfortunately, unlike the first and third product tests that still had very positive points and could be enjoyed by certain consumers, this one should not. Since the objective of this product test was to finalize and optimize the formula created in the second product test, this product test failed on the aspect of flavour, and should be repeated in future when all conditions and requirements can be met, mainly those of the grape must, which means that this product is ideal in the short period following vintage, when the grape must is the freshest. Of course, other factors played in why this product test failed, but this one should be considered vital when attempting to perfect a formula for a grape-based beer. The final product test scored relatively lower on all categories, having the highest percentage of panellists that voted “No” on intent of purchase (86.6 %) as seen in Figure 16. Figure 16. Global appreciation and intent of purchase of the final product test beer based on the number of answers ( n ). Unlike the other products, this attempt however scored more positively on the bitterness (Figure 16) which is an unexpected result as a low amount of bittering hops was used like in the other product tests, and therefore a low bitterness was also present on the final product. Perhaps this could be due to a more confusing flavour and mouthfeel that altered the panellists’ perception of bitterness or at least difficulted the consumer’s ability to perceive the bitterness and may not mean that this product has a better 0 2 4 6 8 10 12 14 16 12345 n Appreciation (1-5) 0 2 4 6 8 10 12 14 Yes No n Answers 52 a risk value of four or higher, as to prevent against dangers which have a high probability of occurrence but low severity, and also, dangers with high severity of occurrence but low probability. The probability of occurrence criteria subdivides into four categories, high (4), where the presence of the identified hazard is certain, being always present, medium-high (3), in which case the presence of the hazard is common, being regularly detected (at least once a year) or the normal practices of the activity can lead to its occurrence, medium (2), when the presence of danger is possible, having occurred in the past on some sporadic situation or there is history or bibliography that supports its presence, and, finally, low (1), where the occurrence is practically impossible or there is minimal presence of danger, having not existed in the past. Table 4. Risk assessment matrix Probability High (4) 4 8 12 16 Medium-High (3) 3 6 9 12 Medium (2) 2 4 6 8 Low (1) 1 2 3 4 Low (1) Medium (2) Medium-High (3) High (4) Severity As for the severity of occurrence, high (4) implies serious health effects, requiring prolonged hospitalization and/or death, medium-high (3) where the effects lead to the affected individual having to be hospitalized, medium (2), in which the effects can be reversed by medical attention, rarely does the affected individual have to be hospitalized, and lastly, low (1), which implies mild severity, and may cause indisposition or discomfort. Based on the 2nd principle, the installation of the HACCP plan makes it possible to identify the stages in which the risk is sufficiently high and in which it will be necessary to use control methods and reduce risk levels to acceptable levels, promoting the implementation of a processing line with controllable risk (Table 5). To identify these CCPs (critical control points), a Decision Tree is used (Figure 22), which, through a sequence of questions about the possible hazards that may occur, allows the detection of the steps where it is necessary to carry out a control in order to reduce or eliminate these same dangers. 53 Table 5. Hazard identification and risk analysis throughout the grape beer production and bottling process Stage Type of Hazard Hazard Risk Control Measures P S R Receipt of raw materials B Presence of mycotoxins or microbial growth 1 3 3 Visual inspection of packages (fungi, moulds, pests, insects). Q Presence of heavy metal residues or pesticides 1 2 2 Analysis of heavy metals and pesticides report sent by the supplier for each batch. F Presence of foreign bodies 1 1 1 Visual inspection of packages. Raw material storage B Microbial growth 1 3 3 Have a stock rotation procedure in place, compliance with hygiene plan, pest control plan and storage procedures. F Presence of pests and insects 1 2 2 Have a stock rotation procedure in place, compliance with hygiene plan, pest control plan and storage procedures. F Cross contamination 1 1 1 Compliance with hygiene plan and storage procedures. Milling F Cross contamination 3 1 3 Compliance with hygiene plan for the equipment and enforce frequent hand hygiene. Brewing B Presence of insects 1 2 2 Visual inspection and compliance with cleaning and sanitation plan. F Presence of foreign bodies 1 1 1 Visual inspection and compliance with cleaning and sanitation plan. Addition of water B Presence of pathogenic microorganisms 1 3 3 Compliance with analysis plan. Q Presence of heavy metals 1 2 2 Reverse osmosis filtration. Boiling B Survival of pathogenic microorganisms 1 4 4 Compliance with manufacturing practices and the hygiene plan for equipment and utensils; control of boiling time and temperature. F Presence of foreign bodies 1 2 2 Compliance with manufacturing practices in place, during utilization of equipment. Visual inspection of ingredients added. Addition of grape must B Presence of pathogenic microorganisms 1 3 3 Regular laboratory analysis and compliance with addition timings. 54 Table 5. Hazard identification and risk analysis throughout the grape beer production and bottling process (cont.) Stage Type of Hazard Hazard Risk Control Measures P S R Cooling Q Cooling chemical contaminants 1 4 4 Compliance with manufacturing practices, visual inspection of equipment, and training employees. Fermentation B Presence of wild yeasts and/or bacteria 1 3 3 Compliance with cleaning and sanitation plan. Q Chemical contaminants (waste from detergents and sanitizers) 1 3 3 Compliance with cleaning and sanitation plan. Dry Hopping B Contamination of beer in fermenter that induces later microbial growth 1 3 3 Compliance with cleaning and sanitation plan as well as good hygiene practices. F Introduction of foreign bodies 1 2 2 Compliance with good manufacturing practices. Filtration B Non-conforming or unsuitable filter 1 1 1 Training employees, compliance with cleaning and sanitation plans. F Filter not compliant. Presence of foreign particles 1 1 1 Training employees, compliance with cleaning and sanitation plans and careful supplier selection. Bottling B Unsanitized bottles that can cause postfilling contamination 1 3 3 Compliance with cleaning and sanitation plan. F Presence of foreign bodies (glass), defective bottles (fractures, chips) 1 4 4 Training employees, compliance with manufacturing practices and work instructions, and careful visual inspection. Shipment (palettizing) F Presence of glass 1 1 1 Visual inspection Finished Product Storage Q Organoleptic changes 3 1 3 Comply with the storage plan (humidity, light, temperature and bottle position) 55 FIGURE 22. HACCP DECISION TREE FOR THE IDENTIFICATION OF CCPS With the aid of the Decision Tree, several critical control points were identified, and are described in Table 6. Next, it is crucial to define the critical limits for these steps, as well as establish monitoring procedures. Critical limits serve as a preventive measure associated with a CCP, corresponding to extreme parameters of acceptability in relation to the safety and quality of the product, being possible values to observe or measure. Each CCP stage will have its critical limits according to possible hazards Q1 Q2 Q3 Q4 56 and control methods. Along with critical limits, it is necessary to define procedures for monitoring the control methods to ensure that the CCP steps are under control. These procedures have a determined frequency, as well as a specific person in charge of monitoring. Following the establishment of critical limits and methods of monitoring these, it is necessary to implement corrective actions for when critical limits are exceeded. In this way, for each hazard of each CCP there must be a specific corrective action, in which the destination of the product that does not comply with the limits of the CCP is indicated. All records must be documented and validated. After establishing corrective actions, procedures for verifying the HACCP system applied must also be stipulated, in which the registration procedures and verification of the validation of the tests, methods and procedures adopted for the monitoring of the CCPs are verified. These verification procedures are performed using an external auditor and may include a review of the plan. In Table 7, the 3rd and 4th principles are applied, presenting critical limits and control methods associated with CCPs, as well as the corrective actions and verification procedures, obtained in accordance with the 5th and 6th HACCP principles. Finally, applying the 7th principle, it will be necessary to develop a system for recording the results of tests carried out for the designated CCPs for which it will be necessary that all documentation related to HACCP is compiled. 57 Table 6. Identification of critical control points throughout grape-based beer production and bottling process Table 7. Establishment of critical limits, corrective measures, monitorization procedures, associated documents and person responsible Stage Type of Hazard Hazard Q1 Q2 Q3 Q4 CCP? Notes Boiling B Survival of pathogenic microorganisms S N N N Cooling Q Cooling chemical contaminants S N S N S CCP1 Bottling F Presence of foreign bodies (glass), defective bottles (fractures, chips) S N S N S CCP2 CCP Stage Critical Limits Corrective Measures Monitorization Associated Documents Responsible CCP1 Cooling Presence of chemical contaminants above permitted limit Discard lots since last ok check of the critical limits, start production of new lots. Maintenance of equipment. Measure of chemical contaminants through refractometer; Frequent substitution of cooling chemicals may imply leaks. Production Manager and Brewer CCP2 Bottling Glass presence detected Reject non-compliant bottles Visual inspection Brewer 58 5. Conclusions The increasing demand of new and differentiated products on the foods and drinks industry gives space to an increase in research and development for the launch of new commercially viable products. One such sector of the drinks industry is the brewing industry, where craft breweries represent the forefront of innovation in terms of new products. Beers that impart characteristics from wine through the use of grape must are a possibility for innovation within the industry and the wine-context of Portugal. With developing an innovative product within these characteristics in mind, the fermentation type that was deemed more suitable for a grape-based beer was the bottom-fermentation, or, a Lager beer where addition of grape must occurs at 50 % of mashing volume. This combination alongside the unmalted cereals for creaminess and citra hops for its aromatic profile make for a very clean base for a grape-based beer, where a better detection of grape must is allowed. However, this is a delicate and complex product subject to adversities and difficult quality control, that is only optimal on a short period after vintage, as the grape must condition, in the amounts used in this study, heavily influences the final product. Other parameters that complicate production of beer with grape must are the difficult conditions it creates for yeast, namely the lower pH and higher alcohol content, which may cause several complications during fermentation. Planning around the nitrogen availability for the yeast is also a crucial factor for this product, and mashing rests should be well defined according to the attenuation level desired. Sensory-wise, aroma and appearance were categories that evaluated positively on most product tests with the addition of grape must to 50 % of mashing volume, but the Grape Lager stood out among the panellists in terms of flavour, which then heavily influences global appreciation and intent of purchase. This beer was the most successful, however, the Grape Weiss also shed an interesting light on the acceptability of Weissbiers, as it draws on many characteristics from them. The statistical analysis conducted pointed to similarities between the Grape Ale and final product test (derived from the Grape Lager), and a certain degree of statistical similarities between the Grape Lager and Grape Weiss. The ones where significant differences were detected were the pairs Grape Ale and Grape Lager, and Grape Lager and the final product test. Nevertheless, positive points were found on all product tests, which further help define a future for a grape-based beer. 59 References Alfeo, V., Todaro, A., Migliore, G., Borsellino, V., Schimmenti, E., (2019). Microbreweries, brewpubs and beerfirms in the Sicilian craft beer industry. International Journal of Wine Business Research , 32(1), 122-138. Alves, V., Gonçalves, J., Figueira, J. A., Ornelas, L. P., Branco, R. N., Câmara, J. S., & Pereira, J. A. 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Retrieved from https://www.hopslist.com/hops/aroma-hops/tettnangtettnanger/ (accessed on August 2022). Hough, J.S., Briggs, D.E., Stevens, R., Young, T.W. (1982). Yeast Growth. In Hough, J. S. (editors) Malting and Brewing Science. Springer. 68 B – Fermentation Profile of the Product Tests Day specific values of temperature, extract concentration ( ºPlato ) and pH for the fermentation and maturation process of the four beers produced, the Grape Ale (Figure B1), Grape Lager (Figure B2), Grape Weiss (Figure B3) and the Final Product Test (Figure B4). Table B1. Variation of temperature ( T ), ºPlato and pH during fermentation of the Grape Ale beer Day T / ºC ºPlato pH 0 40 13.3 4.59 1 40 13.2 3.81 2 40 13.1 3.79 3 40 13.0 3.71 4 40 13.0 3.39 5 23.8 15.0 3.30 6 19.3 14.7 3.34 7 20.7 13.2 3.25 8 18.1 10.5 3.14 10 18.2 8.4 3.26 11 18.7 7.52 3.32 12 19.4 7.5 3.33 14 19.0 7.4 3.36 15 18.0 7.3 3.36 16 18.4 7.2 3.47 17 18.9 7.2 3.38 18 19.1 7.0 3.39 19 19.8 6.9 3.42 Table B2. Variation of temperature ( T ), ºPlato and pH during fermentation of the Grape Lager beer Days T / ºC ºPlato pH 0 13.3 14.6 4.90 1 13.3 14.6 4.89 2 13.0 13.8 4.85 3 12.7 13.8 4.81 7 12.8 9.6 4.47 13 12.5 6.9 4.51 17 15.2 6.8 4.51 20 15.0 6.8 4.53 69 Table B3. Variation of temperature ( T ), ºPlato and pH during fermentation of the Grape Weiss beer Days T / ºC ºPlato pH 0 18 11.1 4.80 1 18 10.75 4.80 2 18 8.26 4.58 5 18 6.81 4.63 6 18 6.81 4.67 7 18 6.81 4.66 10 18 6.62 4.67 11 18 6.62 4.65 12 18 6.62 4.65 Table B4. Variation of temperature ( T ), ºPlato and pH during fermentation of the Final Product Test Days T / ºC ºPlato pH 0 13 14.2 4.9 1 13.5 14 4.95 3 17 8.04 4.97 4 16.5 7.3 5 6 13.5 7.3 5.05 7 19.8 7.6 5.09 8 19.9 7.6 5.07 13 20.0 7.6 5.14 70 C – Statistical Analysis Calculations and Data Statistical calculations and values used to assess differences between products through the Friedman Test and post-hoc analysis Fisher’s Least Significant Difference test. Table C1. Sum, average, standard deviation (SD) and 95 % confidence intervals (95 % CI) for the statistical analysis using Friedman's test A B C D Sum 42 56 49 41 Average 2.8 3.7 3.3 2.7 SD 0.7 1.0 0.7 0.7 95 % CI 0.3 0.5 0.4 0.4 Table C2. Test statistic and tabulated test statistic value for the Friedman’s test T 134.28 χ2α, t -1 7.81 Table C3. Tabulated values for the determination of the LSD rank 1-α z (α/2) 90 % 1.645 95 % 1.96 99 % 2.576 99.9 % 3.291 TABLE C4. Absolute value of the difference between the sum of global appreciation of two products Value in module/absolute A-B 14 A-C 7 A-D 1 B-C 7 B-D 15 C-D 8 Table C5. Median, minimum value, first quartile, third quartile and maximum value for the sensory analysis of the 15 panellists of global appreciation A B C D Median 3 4 3 3 Minimum value 2 2 2 1 First quartile 2 3.5 3 2.5 Third quartile 3 4 4 3 Maximum value 4 5 4 4 71 D – Data on Global Appreciation of the Different Beers Produced for this Study Values of global appreciation, calculated as the average of the five categories assessed in the acceptance test, appearance, aroma, bitterness, flavour and palatefulness. TABLE D1. Global appreciation sensory analysis results for the four product tests Panellist Grape Ale Grape Lager Grape Weiss Final Product Test 1 2 4 4 3 2 3 3 3 2 3 3 4 4 3 4 3 2 2 1 5 3 4 4 3 6 3 4 3 3 7 2 2 2 2 8 4 2 3 2 9 4 4 4 3 10 3 4 4 4 11 3 4 4 3 12 2 5 3 3 13 2 4 3 3 14 2 5 3 3 15 3 5 3 3